Development of Fluorescent Assay for Monitoring of Dehalogenase Activity,Biotechnology Journal

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Development of Fluorescent Assay for Monitoring of Dehalogenase Activity
Biotechnology Journal ( IF 3.2 ) Pub Date : 2018-08-28 , DOI: 10.1002/biot.201800144
Sarka Nevolova ₁ , Elisabet Manaskova ₁ , Stanislav Mazurenko ₁ , Jiri Damborsky _{1,

2,

3} , Zbynek Prokop _{1,

2,

3}

Affiliation

The rapid accumulation of sequence data and powerful protein engineering techniques providing large mutant libraries have greatly heightened interest in efficient methods for biochemical characterization of proteins. Herein is reported a continuous assay for screening of enzymatic activity. The assay is developed and tested with the model enzymes haloalkane dehalogenases and relies upon a fluorescent change of a derivative of 8‐hydroxypyrene‐1,3,6‐trisulphonic acid due to the pH drop associated with the dehalogenation reactions. The assay is performed in a microplate format using a purified enzyme, cell‐free extract or intact cells, making the analysis quick and simple. The method exhibits high sensitivity with a limit of detection of 0.06 mM. The assay is successfully validated with gas chromatography and then applied for screening of 12 haloalkane dehalogenases with the environmental pollutant bis(2‐chloroethyl) ether and chemical warfare agent sulfur mustard. Six enzymes exhibited detectable activity with both substrates. The within‐day variability of the assay for five replicates (n = 5) was 21%.

中文翻译：

监测脱卤素酶活性的荧光检测方法的发展

序列数据的快速积累和强大的蛋白质工程技术提供了大型的突变体文库，极大地提高了人们对蛋白质生物化学表征有效方法的兴趣。本文报道了用于筛选酶活性的连续测定法。该测定方法是使用模型酶卤代烷脱卤酶开发和测试的，并依赖于8-羟基py-1,3,6-三磺酸衍生物的荧光变化，这是由于与脱卤反应相关的pH下降所致。使用纯化的酶，无细胞提取物或完整细胞，以微孔板形式进行测定，从而使分析快速简便。该方法显示出高灵敏度，检测限为0.06 mM。该方法已通过气相色谱法成功验证，然后用于环境污染物双（2-氯乙基）醚和化学战剂硫芥末的筛选，以检测12种卤代烷脱卤酶。两种酶在两种底物上均表现出可检测的活性。五次重复测定的日内变异性（n = 5）为21％。

更新日期：2019-02-26

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